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Nts-polyplex nanoparticles system for gene therapy of cancer

A cancer and compound technology, applied in the manufacture of gene therapy compositions, gene therapy, other methods of inserting foreign genetic materials, etc., can solve problems such as system inappropriateness

Inactive Publication Date: 2013-12-18
CENT DE INVESTIGACION & DE ESTUDIOS AVANZADOS DEL INST POLITECNICO NACIONAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, these systems are not suitable for use in vivo

Method used

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  • Nts-polyplex nanoparticles system for gene therapy of cancer
  • Nts-polyplex nanoparticles system for gene therapy of cancer
  • Nts-polyplex nanoparticles system for gene therapy of cancer

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0095] Example 1.NTS-vector: Synthesis of NTS-(PF)-SPDP-PLL

[0096]For the synthesis of NTS-vectors, we used the following peptides: NTS (sequence ELYENKPRRPYIL), >90% pure, 1,672 Da molecular weight (MM) (Sigma, St. Louis MO, USA); and PF (sequence GLFEAIAEFIEGGWEGLIEGSKKK), 96% pure , 2,695 Da MM (Synpep Corp., Dublín, CA, USA). Both peptides bound simultaneously to PLL (Sigma, St. Louis MO, USA) with MM 25,600-47,900 (mean 36,750 Da). This has 251 potentially reactive amino groups. As a biofunctional crosslinker, we use N-succinimidyl 6-3[3-(2-pyridyldithio)propionamide]hexanoate (LC-SPDP; MM452.52; Cat.21651; Pierce Chemical Co, Rockford, IL, USA).

[0097] NTS-carrier synthesis is a process involving five sequential steps at room temperature: 1) formation of PLL-SPDP conjugate; 2) formation of PLL-SPDP-SH conjugate; 3) formation of NTS-SPDP conjugate; 4) Formation of PFSPDP; and 5) Formation of NTS-carrier [NTS-(PF)-SPDP-PLL] from conjugates previously obtained usi...

Embodiment 2

[0098] Example 2. Formation of PLL-SPDP-SH conjugates

[0099] Dissolve 25 mg of PLL in 2 mL of phosphate buffered saline (PBS: 17.42 mM, deNa 2 HPO 4 , 2.58mM KH 2 HPO 4 , 150mM NaCl, 1mM EDTA, pH7.2). This was mixed with 7.5 mg of LC-SPDP previously dissolved in 30 μL of dimethyl sulfoxide (DMSO). The mixture of PLL and LC-SPDP was incubated for 30 min under continuous agitation. After this time, the resulting conjugate (PLL-SPDP) was purified on an Econo-Pac10DG column (Bio-Rad Laboratories, Hercules, CA, USA) equilibrated with PBS. We collected fractions of 1 mL. 3 μl aliquots were taken from each fraction and read in a NanoDrop spectrophotometer (NanoDrop Technologies ND_1000) to determine absorbance at 215 and 280 nm.

[0100] The Econo-Pac10DG column allows molecules <6,000 Da to be eluted; thus, the PLL-SPDP conjugate (52,043 Da) is obtained in the first peak (3-7 mL) and released in the second peak (8-13 mL). Free SPDP (425.5 Da) and N-hydroxysuccinimide (114 ...

Embodiment 3

[0102] Example 3. Formation of NT-SPDP conjugates

[0103] 10 mg of NTS was dissolved in 2 mL of PBS and mixed with 5 mg of LC-SPDP previously dissolved in 30 μL of DMSO. The reaction mixture was incubated for 30 minutes with constant stirring. After incubation, the NTS-SPDP conjugate was purified on a Sephadex G-10 column (Pharmacia Fine Chemicals AB, Uppsala, Sweden) equilibrated with PBS. 0.1 mL fractions were collected; 3 μl aliquots of each fraction were read on the NanoDrop to determine their absorbance at 215 and 280 nm. Sephadex G-10 has the ability to avoid <700 Da; thus, the NTS-SPDP conjugate (2,419 Da) elutes in the first peak volume (3.5-8.4 mL). Fractions corresponding to the first peak were collected and concentrated to 1 mL.

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Abstract

The present invention describes a system of gene carrier nanoparticles capable of specifically internalize into cancer cells, eg, cancer cells involved in breast cancer, in vitro and in vivo. The system described allows the introduction of therapeutic genes specifically into target cells through NSTR1 receptor-mediated endocytosis of said system, making it possible to provide treatment for this type of conditions, for example by systemic, intravenous, or in situ administration.

Description

technical field [0001] The present invention relates to the field of antitumor gene therapy, which involves the specific introduction of functional genes into cancer cell types. More specifically, the present invention relates to strategies for initiating the transfer of genetic material in vivo by using nanoparticles carrying a gene that is expressed in response to therapeutic purposes and internalized in, for example, breast cancer cells to prevent disease progress and transfer. Background technique [0002] Breast cancer is the leading cause of death in women worldwide. It is estimated that one in nine women who live to age 85 will develop the condition at some point in their lives. Breast cancer exhibits higher rates in developed countries, although breast cancer rates are also rising in developing countries, making this difference narrowing. According to the World Health Organization (WHO), the United States, France, Iceland, Great Britain, Canada, other European cou...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K48/00C07K7/08C12N9/12A61K47/48
CPCC12N9/1211C12Y207/01021C07K2319/035A61K48/0025A61K48/0091C12N15/87A61K47/62A61K47/645A61K47/6929A61K38/10A61K48/00C07K7/083
Inventor R·A·卡斯蒂略罗德里格斯M·L·埃斯科韦多桑切斯D·马丁内斯丰
Owner CENT DE INVESTIGACION & DE ESTUDIOS AVANZADOS DEL INST POLITECNICO NACIONAL
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